Improved Screening of cDNAs Generated by mRNA Differential Display Enables the Selection of True Positives and the Isolation of Weakly Expressed Messages

The high percentage of false positives generated by differential display (as high as 85%) has previously limited the potential of the method. This report describes an efficient methodology that enables false positives to be discarded prior to cloning, via reverse Northern analysis. This first step of the screening also allows the detection of putative lowabundance differential clones. Following cloning, a second reverseNorthern combined with partial DNA sequencing and RT-PCR detection allows isolation of all differential cDNAs including very lowabundance clones. Use of the sequential screening procedure described here led to the isolation of novel tomato genes responding to the plant hormone ethylene while minimising labor and materials input

ER5, a tomato cDNA encoding an ethylene-responsive LEA-like protein: characterization and expression in response to drought, ABA and wounding

We report the isolation by differential display of a novel tomato ethylene-responsive cDNA, designated ER5. RT-PCR analysis of ER5 expression revealed an early (15 min) and transient induction by ethylene in tomato fruit, leaves and roots. ER5 mRNA accumulated during 2 h of ethylene treatment and thereafter underwent a dramatic decline leading to undetectable expression after 5 h of treatment. The full-length cDNA clone of 748 bp was obtained and DNA sequence analysis showed strong homologies to members of the atypical hydrophobic group of the LEA protein family. The predicted amino acid sequence shows 67%, 64%, 64%, and 61%sequence identity with the tomato Lemmi9, soybean D95-4, cotton Lea14-A, and resurrection plant pcC27-45 gene products, respectively. As with the other members of this group, ER5 encodes a predominantly hydrophobic protein. Prolonged drought stress stimulates ER5 expression in leaves and roots, while ABA induction of this ethylene-responsive clone is confined to the leaves. The use of 1-MCP, an inhibitor of ethylene action, indicates that the drought induction of ER5 is ethylene-mediated in tomato roots. Finally, wounding stimulates ER5 mRNA accumulation in leaves and roots. Among the Lea gene family this novel clone is the first to display an ethylene-regulated expression

Recent Developments on the Role of Ethylene in the Ripening of Climacteric Fruit

It has long been recognised that ethylene plays a major role in the ripening process of climacteric fruit. A more thorough analysis, however, has revealed that a number of biochemical and molecular processes associated with climacteric fruit ripening are ethylene-independent. One of the crucial steps of the onset of ripening is the induction of autocatalytic ethylene production. In ethylene-suppressed melons, ACC synthase activity is induced at the same time as in control melons, indicating that ACC biosynthesis during the early stages of ripening seems to be a developmentally-regulated (ethylene-independent) process. The various ripening events exhibit differential sensitivity to ethylene. For instance, the threshold level for degreening of the rind is 1ppm, while 2.5 ppm are required to trigger some components of the softening process. The saturating level of ethylene producing maximum effects is less than 5 ppm, which is by far lower than the internal ethylene concentrations found in the fruit at the climacteric peak (over 100 ppm). In many fruit chilling temperatures hasten ethylene production and ripening and in some late season pear varieties, exposure to chilling temperatures is even absolutely required for the attainment of the capacity to synthesize autocatalytic ethylene. This is correlated with the stimulation of expression of ACC oxidase and of members of the ACC synthase gene family. Ethylene operates via a perception and transduction pathway to induce the expression of genes responsible for the biochemical and physiological changes observed during ripening. However, only a few genes induced via the ethylene transduction pathway have been described so far. We have used a differential display method to isolate novel ethylene-reponsive (ER) cDNA clones of tomato that potentially play a role in propagating the ethylene response and in regulating fruit ripening. Collectively, these data permit a general scheme of the molecular mechanisms of fruit ripening to be proposed

Tomato EF-Tsmt, a functional mitochondrial translation elongation factor from higher plants

Ethylene-induced ripening in tomato (Lycopersicon esculentum) resulted in the accumulation of a transcript designated LeEF-Tsmt that encodes a protein with significant homology to bacterial Ts translational elongation factor (EF-Ts). Transient expression in tobacco and sunflower protoplasts of full-length and truncated LeEF-Tsmt- GFP fusion constructs and confocal microscopy observations clearly demonstrated the targeting of LeEF-Tsmt to mitochondria and not to chloroplasts and the requirement for a signal peptide for the proper sorting of the protein. Escherichia coli recombinant LeEF-Tsmt co-eluted from Ni-NTA resins with a protein corresponding to the molecular weight of the elongation factor EF-Tu of E. coli, indicating an interaction with bacterial EF-Tu. Increasing the GDP concentration in the extraction buffer reduced the amount of EF-Tu in the purified LeEF-Tsmt fraction. The purified LeEF-Tsmt stimulated the poly(U)-directed polymerization of phenylalanine 10-fold in the presence of EF-Tu. Furthermore, LeEF-Tsmt was capable of catalysing the nucleotide exchange reaction with E. coli EF-Tu. Altogether, these data demonstrate that LeEF-Tsmt encodes a functional mitochondrial EF-Ts. LeEFTsmt represents the first mitochondrial elongation factor to be isolated and functionally characterized in higher plants

Modeling Subjective Symptoms Related to Micro-Hydrargyrism in a Population of Moroccan Dentists

BACKGROUND፡ The ability of mercury to deposit throughout the body and alter a wide range of molecular and cellular pathways results in a polymorphic and complex clinical phenotype with over 250 possible symptoms. However, some of them are recurrently cited as evoking chronic mercury poisoning. In this light, dentists users of dental amalgams are chronically exposed to mercury so that in-depth epidemiological investigations and adapted statistical methods are required to highlight adverse effects of this exposure.METHODS: In order to study the health impact of the occupational mercury exposure in a population of liberal dentists practicing in two Moroccan regions, a list of eighteen subjective symptoms commonly associated with micro-hydrargyrism was drawn up. Then, seven statisctical models adapted to count data were fitted. Finally, three methods were used to compare their relative performance in order to choose the most appropriate one.RESULTS: The adopted logical path, from the response variable selection till models’ comparison, led us to lean towards quasi- Poisson regression as the best way to predict the number of symptoms declared by dentists according to mercury exposure.CONCLUSIONS: Interpretation of the selected model allowed us to conclude that the reduction of dental amalgam use allows the reduction of subjective symptoms related to mercury exposure

Time-resolved FRET reports FGFR1 dimerization and formation of a complex with its effector PLCγ1.

In vitro and in vivo imaging of protein tyrosine kinase activity requires minimally invasive, molecularly precise optical probes to provide spatiotemporal mechanistic information of dimerization and complex formation with downstream effectors. We present here a construct with genetically encoded, site-specifically incorporated, bioorthogonal reporter that can be selectively labelled with exogenous fluorogenic probes to monitor the structure and function of fibroblast growth factor receptor (FGFR). GyrB.FGFR1KD.TC contains a coumermycin-induced artificial dimerizer (GyrB), FGFR1 kinase domain (KD) and a tetracysteine (TC) motif that enables fluorescent labelling with biarsenical dyes FlAsH-EDT2 and ReAsH-EDT2. We generated bimolecular system for time-resolved FRET (TR-FRET) studies, which pairs FlAsH-tagged GyrB.FGFR1KD.TC and N-terminal Src homology 2 (nSH2) domain of phospholipase Cγ (PLCγ), a downstream effector of FGFR1, fused to mTurquoise fluorescent protein (mTFP). We demonstrated phosphorylation-dependent TR-FRET readout of complex formation between mTFP.nSH2 and GyrB.FGFR1KD.TC. By further application of TR-FRET, we also demonstrated formation of the GyrB.FGFR1KD.TC homodimer by coumermycin-induced dimerization. Herein, we present a spectroscopic FRET approach to facilitate and propagate studies that would provide structural and functional insights for FGFR and other tyrosine kinases

Refrigeration for preservation of the Moroccan date: Situation and physical analysis of the quality

La production des dattes au Maroc, estimée à près de cent mille tonnes par année favorable, contribue à la génération des revenus, à hauteur de 40 à 60 % pour près de 2 millions de la population oasienne. Cependant, cette production dattière est exposée à des contraintes liées aux bonnes pratiques de récolte, de traitements post-récolte, de conservation et de stockage. Cette étude consiste en un diagnostic (basé sur des enquêtes) des conditions de traitement et de stockage des dattes dans 5 entrepôts frigorifiques installés dans les oasis du Sud-est Marocain. Ces enquêtes ont été complétées par un suivi, au cours du stockage, de la qualité et du taux de déshydratation de deux principales variétés nobles de dattes: Majhoul et Boufeggous. Les résultats des enquêtes ont montré que la capacité de stockage de la majorité des entrepôts est de l’ordre de 400 tonnes. Le traitement des dattes se fait en plusieurs étapes dont les plus importantes sont la fumigation à la phosphine (PH3) pendant 3 à 5 jours à température ambiante et le triage des dattes en trois catégories, en fonction de leur calibre et de la présence de défauts. Après ces traitements, les dattes sont stockées, toutes variétés confondues, dans la même chambre froide à une température variant de 0 à 4°C pendant une période allant de 1 à 10 mois. Les chambres de stockage ne sont pas équipées d’humidificateurs, ce qui ne permet pas une maîtrise de l’humidité relative. Les défauts majeurs observés au cours du stockage sont l’apparition des taches glucosées à l’intérieur de l’épiderme des fruits (57 % en moyenne) et le détachement de l’épicarpe des dattes (20 % en moyenne). La cinétique de déshydratation a montré une similitude entre les variétés avec une moyenne respective, de la perte en eau, de 6,5 % pour la variété Majhoul et 7 % pour la variété Boufeggous après 12 mois du stockage. Mots clés : Maroc, datte, stockage frigorifique, qualité, déshydratation The production of date palm in Morocco, estimated at around one hundred thousand tons per year, contributes to the generation of income, 40 to 60 %, for nearly 2 million of the oasis population. However, this date palm production is exposed to constraints related to good harvesting practices, post-harvest treatments, conservation and storage. This study consists of a diagnosis (based on surveys) of date processing and storage conditions in five cold storage units located in the oases of South-East of Morocco. These surveys were supplemented by monitoring the quality and dehydration rate of two main varieties of date-palm fruits: Majhoul and Boufeggous. Survey results showed that the storage capacity of the majority of cold storage units is about 400 tons. The date-palm fruits are processed in several stages; the most important of which are fumigation with phosphine (PH3) for 3 to 5 days at room temperature and manual sorting of dates into three categories according to their size and the presence of defects. After these treatments, all varieties of dates are stored in the same cold room at a temperature of 0 to 4°C for a period of 1 to 10 months. The storage rooms are not equipped with humidifiers which do not allow a control of the relative humidity. The major defects observed during storage are the appearance of glucose spots inside the fruit epidermis (57 % on average) and the detachment of the date-palm fruit wall (20 % on average). The kinetics of dehydration showed a similarity between the varieties with a respective average of 6.5 % for the Majhoul and 7 % for Boufeggous, after 12 months of storage. Key-Words: Morocco, date, cold storage, quality, dehydration. &nbsp